Use of Soluble Fgl2 As An Immunosuppressant

ABSTRACT

Methods and compositions for inducing immune suppression are disclosed. The methods involve administering an effective amount of a soluble fgl2 protein or a nucleic acid encoding a soluble fgl2 protein. The methods are useful in preventing graft rejection, autoimmune disease, and allergies.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for modulatingan immune response. Specifically, the invention includes the use of thesoluble fgl2 protein to suppress an immune response.

BACKGROUND OF THE INVENTION

Proteins homologous to the carboxyl terminus of the β and γ chains(fibrinogen-related domain or FRED²) of fibrinogen, includingangiopoietins, ficolins and tenascins, have been classified into thefibrinogen-related protein superfamily and have been demonstrated toexert multifaceted roles in immune responses (1-3). For example,fibrinogen can act as a “bridge” between α_(m)β2-bearing leukocytes toICAM-1 on endothelial cells, and the engagement of α_(m)β2 by fibrinogentriggers a series of intracellular signaling events and cellularresponses including cytokine secretion and nuclear factor-κB activation(4, 5). Angiopoietin-1 inhibits endothelial cell permeability inresponse to thrombin and vascular endothelial growth factor in vitro viathe regulation of the junctional complexes, PECAM-1 and vascularendothelial cadherin (6). Two independent studies have reported thatsoluble tenascin blocks T cell activation induced by soluble antigens,alloantigens, or the mitogen Con A (7, 8).

Fgl2, also known as fibroleukin, has been demonstrated to be involved inthe pathogenesis of diseases including viral-induced fulminant hepatitisand Th1 cytokine-induced fetal loss syndrome, in which fibrin depositionis a prominent feature (9-11). The gene fgl2 was originally cloned fromCTL and the encoded protein shares a 36% homology to the fibrinogen βand γ chains and a 40% homology to the FRED of tenascin (1, 12). Thecoagulation activity of fgl2 was first described in a murine fulminanthepatitis model (13, 14) and fgl2 prothrombinase was detected inactivated reticuloendothelial cells (macrophages and endothelial cells)(9, 15, 16). Fgl2 functions as an immune coagulant with the ability togenerate thrombin directly, and thus, fgl2 appears to play an importantrole in innate immunity.

Human fgl2/fibroleukin expressed by peripheral blood CD4⁺ and CD8⁺T-cells has been shown to be a secreted protein devoid of coagulationactivity (17, 18). However, the function for soluble fgl2 proteingenerated by T-cells has herebefore remained undefined.

SUMMARY OF THE INVENTION

The present inventor has demonstrated that soluble fgl2 protein inhibitsT-cell proliferation induced by alloantigen, anti-CD3/anti-CD28 mAbs andConcanavalin-A (ConA) in a dose-and time-dependent manner. Promotion ofa Th2 cytokine profile was observed in a fgl2-treated allogeneicresponse. In addition, fgl2 protein abrogated LPS-induced maturation ofbone marrow-derived dendritic cells (DC), resulting in a reduced abilityto induce alloreactive T cell proliferation. Further, in a rat to mouseskingraft xenotransplantation model, soluble fgl2 protein exhibitsimmunosuppressive properties as shown by the inhibition of T cellsproliferation in a one way xeno-mixed lymphocyte reaction. All of theseresults indicate that soluble fgl2 is an effective immune suppressantand soluble fgl2 has properties distinct from the prothrombinaseactivity of membrane bound fgl2.

Consequently, the present invention provides a method of suppressing animmune response comprising administering an effective amount of asoluble fgl2 protein or a nucleic acid sequence encoding a soluble fgl2protein to an animal in need of such treatment. The invention alsoincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid sequence encoding a soluble fgl2 protein to suppress animmune response or in the manufacture of a medicament to suppress animmune response.

In one embodiment, the present invention provides a method of preventingor inhibiting T-cell proliferation and/or DC maturation comprisingadministering an effective amount of a soluble fgl2 protein or a nucleicacid sequence encoding a soluble fgl2 protein to an animal in need ofsuch treatment. The invention includes a use of an effective amount of asoluble fgl2 protein or a nucleic acid sequence encoding a soluble fgl2protein to prevent or inhibit T-cell proliferation and/or DC maturationor in the manufacture of a medicament to prevent or inhibit T cellproliferation and/or DC maturation.

In a further embodiment, the present invention provides a method ofpromoting a Th2 cytokine response comprising administering an effectiveamount of a soluble fgl2 protein or a nucleic acid sequence encoding asoluble fgl2 protein to an animal in need thereof. The inventionincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid sequence encoding a soluble fgl2 protein to promote a Th2cytokine response or in the manufacture of a medicament to promote a Th2cytokine response.

The present invention further provides a method of treating a disease orcondition wherein it is desirable to suppress an immune responsecomprising administering an effective amount of a soluble fgl2 proteinor a nucleic acid sequence encoding a soluble fgl2 protein to an animalin need of such treatment. The invention also includes a use of aneffective amount of a soluble fgl2 protein or a nucleic acid sequenceencoding a soluble fgl2 protein to treat a disease or condition whereinit is desirable to suppress an immune response or in the manufacture ofa medicament to suppress an immune response.

The method of the invention may be used to treat any disease orcondition wherein it is desirable to suppress an immune response, forexample, to induce tolerance to transplanted organs or tissues, treatinggraft versus host disease, treating autoimmune diseases and treatingallergies.

The invention also includes pharmaceutical compositions containingsoluble fgl2 proteins or nucleic acids encoding soluble fgl2 proteinsfor use in suppressing an immune response.

The present invention also provides an antibody, preferably a monoclonalantibody, to soluble fgl2. In one embodiment, said monoclonal antibodybinds proximate to the cleavage site of soluble fgl2 (e.g. the cleavagesite of membrane/soluble fgl2).

The invention further provides a method for diagnosing a conditionrelated to soluble fgl2 expression or activity. In one embodiment, saidmethod comprises obtaining a biological sample from a patient (such as ablood or tissue sample) and incubating said sample with an antibody forsoluble fgl2, preferably a monoclonal antibody, under conditions thatpermit formation of a soluble fgl2/antibody complex. Said method permitsthe detection and/or determination of the presence and or level ofsoluble fgl2 in the sample, the presence or particular level of solublefgl2 being indicative of a soluble fgl2 related condition. In anotherembodiment, said antibody is a labelled antibody. In another embodiment,the amount of soluble fgl2 is determined by the amount of complexedsoluble fgl2 with said soluble fgl2, either directly or indirectly. Forinstance, if a particular amount of antibody is used, then the amount ofcomplexed or remaining uncomplexed (or free) antibody can be measured toinfer the amount of soluble fgl2 present in the sample.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIGS. 1A and B shows the generation of soluble fgl2 protein in abaculovirus system. SDS-PAGE followed by Coomassie blue staining of fgl2protein purified from recombinant baculovirus-infected High 5 (H5) cellsshowed a dominant band at 65-kDa confirmed by (b) Western blotting. Fgl2protein was generated using a baculovirus expression system and purifiedas described in the “Materials and Methods”. Cell lysates prepared underdenaturing condition were incubated with Ni-NTA resin and proteins boundto Ni-NTA resin were eluted. All eluted proteins (10 μg) were analyzedon 10% SDS-PAGE followed by (a) Coomassie blue staining or (b) Westernblot analysis probed with polyclonal rabbit anti-mouse fgl2 IgG asdescribed in the “Materials and Methods”. Lane 1, eluted proteins fromlysate of the uninfected H5 cells; lane 2, eluted proteins from lysateof the wild type baculovirus infected H5 cells; lane 3, eluted proteinsfrom lysate of the recombinant baculovirus-infected H5 cells.

FIGS. 2A and B shows the binding of biotinylated-soluble fgl2 to T cellsand BM-derived LPS-induced mature DC. T cells and BM-derived LPS-inducedmature DC cells were prepared. CD3- and CD11c-positive populations weregated and the binding of soluble fgl2 to T cells (a) and dendritic cells(b) was determined as described in the “Materials and Methods” (shown ingrey area). The white area shows the binding of biotinylated-BSAcontrol.

FIG. 3A-C shows the inhibitory effects of soluble fgl2 protein on T cellproliferation induced by different stimuli. Effects of soluble fgl2protein (shown in solid line) and human fibrinogen (shown in dottedline) on T cell proliferation induced by (a) alloantigens, (b)immobilized anti-CD3 mAb (1 μg/ml) with soluble anti-CD28 mAb (20 ng/ml)and (c) Con A (5 μg/ml) with various concentrations of soluble fgl2 orhuman fibrinogen added at the initiation of cultures as described in the“Materials and Methods”. Proliferation was determined by [³H]thymidineuptake in triplicate wells and the incorporation in fgl2-untreatedcontrol cultures (shown as 0 ng/ml soluble fgl2 protein added) was asfollows: (a) 14,892±978 cpm; (b) 16,890±1146 cpm; (c) 39,368±3560 cpm,with background 595±193 cpm. The data are expressed as percentageinhibition (soluble fgl2-untreated cultures shown as 0% inhibition) andare representative of three separate experiments. **, p<0.01 and *,p<0.05 compared with control groups (far left, no soluble fgl2 wasadded).

FIGS. 4A and B shows the inhibitory effect of soluble fgl2 onalloreactive T cell proliferation. (a) Kinetics of the effect of solublefgl2 on alloreactive T cell proliferation soluble fgl2 (1 μg/ml) wasadded on d 0 to d 3 to an ongoing allogeneic reaction as described inthe “Materials and Methods”. Proliferation was determined by[³H]thymidine uptake in triplicate wells and the incorporation insoluble fgl2-untreated control cultures (shown as Bc+AJ) was: 14,892±978cpm with background 523±102 cpm. The data are expressed as percentageinhibition (soluble fgl2-untreated cultures shown as 0% inhibition) andare representative of three separate experiments. **, p<0.01 and *,p<0.05 compared with control groups for both % suppression and cpm (farleft, no soluble fgl2 was added). (b) The inhibitory effect of solublefgl2 protein on alloreactive T cell proliferation was neutralized by ananti-mouse fgl2 mAb. One-way MLC was set up as described previously anda monoclonal anti-mouse fgl2 Ab (1 μg) was added at the beginning ofcultures along with the addition of soluble fgl2 protein (1 μg/ml). Anisotype control mAb was added for comparison. **, p<0.01 compared withcontrol groups (second left, no anti-mouse fgl2 Ab added).

FIG. 5A-C shows soluble fgl2 protein (1 μg/ml) inhibited MLR withpromotion of a Th2 cytokine profile. (a) Lymph node T cells wereco-cultured with allogeneic BM-derived DC as described in the “Materialsand Methods”. soluble fgl2 protein (1 ng/ml to 1 μg/ml) was added to theculture at the beginning (d 0) and cell proliferation was measured using[³H] thymidine as described. The incorporation of [³H] thymidine insoluble fgl2-untreated control cultures (shown as 0 ng/ml soluble fgl2protein added) was: 5,546±620 cpm with background 355±55 cpm. The dataare expressed as percentage inhibition (fgl2-untreated cultures shown as0% inhibition). (b & c) Supernatants from the allogeneic cultures in theabsence (open bar) or presence of 1 μg/ml soluble fgl2 protein (closedbar) or 1 ng/ml soluble fgl2 protein (hatched bar) were collected at 24h to measure the levels of cytokine produced. The data arerepresentative of three separate experiments. **, p<0.01 and *, p<0.05compared with control groups (far left, no soluble fgl2 was added).

FIG. 6A-F shows cell surface phenotype of DC generated in the absence(control) or presence of soluble fgl2 (1 μg/ml) during maturation. BMcells were prepared and cultured for 7 d in the presence of GM-CSF andIL-4 to derive immature DC as described in the “Materials and Methods”.Immature DCs were stimulated with LPS (200 ng/ml) to reach finalmaturation for 2 days in the absence and presence of soluble fgl2protein. The expression of surface molecules including CD11c, CD80,CD86, MHC class I and class II and CD40 were measured by flow cytometryanalysis. Results are representative of 3 independent experiments. Graylines show fluorescence signals of cells treated with soluble fgl2, andstained with the specific Abs. Black lines represent non-solublefgl2-treated cells. The blue shaded areas are the appropriateisotype-matched Ig control.

FIG. 7 shows DC treated with soluble fgl2 during LPS-induced maturationexhibited impaired ability to induce allogeneic responses. DC treatedwith soluble fgl2 (1 μg/ml) during LPS-induced maturation (DCFgl2) and Tcells treated with soluble fgl2 protein (1 μg/ml) prior culturing withDC (TFgl2) were used to examine their ability to induce allogeneicresponse as described in the “Materials and Methods”. In some cases, DCtreated with soluble fgl2 during maturation (DCFgl2) were employed toactivate allogeneic T cell proliferation with soluble fgl2 protein (1μg/ml) added to the culture (last column). Proliferation was determinedby [³H] thymidine uptake in triplicate wells and the uptake in untreatedcultures (shown as T+DC) was: 5,546±620 cpm with background 355±55 cpm.The data are representative of three separate experiments. **, p<0.01and *, p<0.05 compared with control groups (second left, no soluble fgl2was added).

FIGS. 8A and B shows the effect of soluble fgl2 on translocation ofNF-κB in LPS Stimulated Dendritic Cells. A, Dual immunofluorescencestaining of LPS and soluble fgl2+LPS treated BM-derived DC. After 1 h ofexposure to LPS or soluble fgl2+LPS immunostaining was carried out asdescribed in the material and methods. Each Panel (a-f) shows the samecells visualized by immunofluorescence using different filters. Cells in(a) and (d) show red fluorescence anti-p65 staining. Cells in (b) and(e) show blue chromosomal DAPI staining. Panels (c) and (f) show anoverlay of the anti-p65 and DAPI staining. The purple color representscells that have p65 translocation. LPS exposed cells had marked NF-κBtranslocation shown by the accumulation of p65 in the nucleus, confirmedby DAPI nuclear staining (c). Incubation with soluble fgl2 during LPSexposure was able to inhibit NF-κB translocation shown by diffuse p65staining and distinct blue of the DAPI staining in most of cells (f). B,Histogram showing the percent p65 NF-κB translocation in at T=1 h afterLPS stimulation in soluble fgl2 treated and untreated groups. An averageof 100 cells were counted from greater than 3 different fields. There isa significant reduction in p65 NF-κB translocation with soluble fgl2treatment (p<0.01).

FIG. 9 is a graph showing the effects of fgl2 protein on T cellproliferation in a one-way xenogeneic mixed lymphocyte reaction. Fgl2protein inhibited the proliferation of primed T cells re-challenged withthe splenic cells of the Wistar rat origin in a dose-dependent manner.Proliferation was determined by [³H] thymidine uptake in triplicatewells. The data are expressed as percentage of inhibition(fgl2-untreated samples are shown as 0% of inhibition) and arerepresentative of three separate experiments. Statistical analysis wasperformed using the Student-Newman-Keuls method or one-way ANOVA. *,p<0.05 and **, p<0.02 compared with control groups (no fgl2 added).

FIG. 10 shows 5′RACE analysis of fgl2 gene expression. Full length mRNAfgl2 transcripts were detected in na{dot over (i)}ve T cells (A), Bcells stimulated with 10 mg/ml LPS for 3 days (B), immature and maturedendritic cells (C), na{dot over (i)}ve macrophages, and macrophagesstimulated with 100 U/ml IFN-γ for 24 hrs (D), whereas fgl2 was notdetected in naive B cells (B). Three shorter fgl2 mRNA transcripts,corresponding to molecular sizes 1.2 kb, 1.0 kb and 0.8 kb were detectedin T cells stimulated with 5 μg/ml Con A for 3 days (A).

FIG. 11 shows the sequence analysis of the 5′RACE products. Thesequences of the 1.3 kb band observed in the 5′RACE product of theantigen presenting cells and na{dot over (i)}ve T cells corresponded tothe full length fgl2 mRNA and the resulting protein with theprothrombinase activity. The nucleotide position of the ATG translationstart codon of the full length fgl2 mRNA is referred to as +1. Thesequences of three short fgl2 mRNA transcripts seen in Con-A stimulatedT cells indicated they were the result of alternative transcriptionalstart sites and not alternative splicing at the exon-intron junction.The longest open reading frames of the 1.2 kb, 1.0 kb and 0.8 kbfragments as determined by the first methionine codon with a Kozakconsensus sequence would begin translation at the ATG nucleotideposition +217, +475, and +577, respectively.

FIGS. 12A and B shows the nucleic acid sequence (FIG. 12A and SEQ IDNO:1) encoding truncation fragment 1 and the amino acid sequence oftruncation fragment 1 (FIG. 12B and SEQ ID NO:2).

FIGS. 13A and B shows the nucleic acid sequence (FIG. 13A and SEQ IDNO:3) encoding truncation fragment 2 and the amino acid sequence oftruncation fragment 2 (FIG. 13B and SEQ ID NO:4).

FIGS. 14A and B shows the nucleic acid sequence (FIG. 14A and SEQ IDNO:5) encoding truncation fragment 3 and the amino acid sequence oftruncation fragment 3 (FIG. 14B and SEQ ID NO:6).

DETAILED DESCRIPTION OF THE INVENTION

The present inventor has provided the first evidence that soluble fgl2protein exhibits immunomodulatory properties with suppressive effects onT-cell proliferation and DC maturation. Promotion of a Th2 cytokineresponse has been shown to improve transplant survival. The inventor hasshown that addition of soluble fgl2 protein in a mixed leukocytereaction resulted in an increased production of IL-4 and IL-10 and adecreased production of IL-2 and IFN-γ. The inventor has also shown thatsoluble fgl2 inhibits T cell proliferation in a one way xeno-mixedlymphocyte reaction in a rat to mouse skin graft xenotransplantationmodel.

DCs are professional antigen presenting cells (APCs) which exhibit aunique ability to stimulate both naive and memory T-lymphocytes. Theirpotential to determine the balance between immunity and tolerance allowsDCs to be a target for therapeutic manipulation of immune responsesagainst alloantigens. It has been reported that bone marrow-derivedimmature DCs can induce hyper-responsiveness in allogeneic T-cells andsignificantly prolong cardiac allograft survival when injected intorecipient mice (25). Herein it is demonstrated that soluble fgl2prevented full maturation of DCs by lowering the expression levels ofCD80^(hi), CD86^(hi) and MHC class II^(hi). No significant effects onMHC class I, CD11c and CD40 expression were observed.

The present inventor has demonstrated that soluble fgl2 protein exhibitsimmunomodulatory properties with no direct prothrombinase activity. Thefindings that soluble fgl2 inhibited T-cell proliferation, promoted Th2cytokine expression and prevented DC maturation suggest a novel usage ofsoluble fgl2 protein as an immunosuppressive agent which may induceT-cell tolerance and therefore improve graft survival.

I. Soluble Fgl2

As the present inventor was the first to identify the immunosuppressiveactivity of soluble fgl2, the present invention relates to all such usesof soluble fgl2 including the therapeutic and diagnostic methods andpharmaceutical compositions which are described herein below.

In all embodiments of the invention, the term “soluble fgl2 protein”means a non-membrane bound fgl2 protein including soluble fgl2 from anyspecies or source and includes analogs and fragments or portions of asoluble fgl2 protein. Soluble fgl2 proteins (or analogs, fragments orportions thereof) of the invention are those that are able to suppressan immune response and preferably inhibit T cell proliferation. Thesoluble fgl2 proteins may have no prothrombinase activity.

Determining whether a particular soluble fgl2 protein can suppress animmune response can be assessed using known in vitro immune assaysincluding, but not limited to, inhibiting a mixed leucocyte reaction;inhibiting T-cell proliferation; inhibiting interleukin-2 production;inhibiting IFNγ production; inhibiting a Th1 cytokine profile; inducingIL-4 production; inducing TGFβ production; inducing IL-10 production;inducing a Th2 cytokine profile; inhibiting immunoglobulin production;altering serum immunoglobulin isotype profiles (from those associatedwith Th1 type immunity—e.g. in the mouse, IgG1 and IgG2a, to thoseassociated with Th2 type immunity—e.g. in the mouse, IgG2b, IgG3);inhibition of dendritic cell maturation; and any other assay that wouldbe known to one of skill in the art to be useful in detecting immunesuppression.

The soluble fgl2 protein may be obtained from known sources or preparedusing known techniques such as recombinant or synthetic technology. Theprotein may have any of the known published sequences for fgl2 which canbe obtained from public sources such as GenBank. Examples of suchsequences include, but are not limited to Accession Nos. AAL68855;P12804; Q14314; NP032039; AAG42269; AAD10825; AAB88815; AAB88814;NP006673; AAC16423; AAC16422; AAB92553. The fgl2 sequences can also befound in WO 98/51335 (published Nov. 19, 1998) and in Marazzi et al.(1998), Rüegg et al. (1995) and Yuwaraj et al. (2001) (17, 18, 41)). Theaforementioned sequences are incorporated herein by reference. Thesoluble fgl2 protein can be obtained from any species, preferably amammal including human and mouse.

The term “analog” as used herein includes any peptide having an aminoacid sequence that is similar to any of the known soluble fgl2 sequencesin which one or more residues have been substituted with a functionallysimilar residue and which displays the ability to suppress an immuneresponse. Analogs include conservative substitutions, derivatives,peptide mimetics and homologs of soluble fgl2. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as alanine, isoleucine, valine, leucine or methionine foranother, the substitution of one polar (hydrophilic) residue for anothersuch as between arginine and lysine, between glutamine and asparagine,between glycine and serine, the substitution of one basic residue suchas lysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another. Thephrase “conservative substitution” also includes the use of a chemicallyderivatized residue in place of a non-derivatized residue provided thatsuch polypeptide displays the requisite activity.

The term “derivative” as used herein refers to a peptide having one ormore residues chemically derivatized by reaction of a functional sidegroup. Such derivatized molecules include for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as derivatives are those peptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For examples: 4-hydroxyproline may be substituted forproline; 5 hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

The term “peptide mimetic” as used herein includes synthetic structureswhich may or may not contain amino acids and/or peptide bonds but retainthe structural and functional features of a soluble fgl2 protein orpeptide. Peptide mimetics also include peptoids, oligopeptoids; andpeptide libraries containing peptides of a designed length representingall possible sequences of amino acids corresponding to a soluble fgl2protein. Peptide mimetics may be designed based on information obtainedby systematic replacement of L-amino acids by D-amino acids, replacementof side chains with groups having different electronic properties, andby systematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

The soluble fgl2 protein may be modified to make it more therapeuticallyeffective or suitable. For example, the soluble fgl2 protein may becyclized as cyclization allows a peptide to assume a more favourableconformation. Cyclization of the soluble fgl2 peptides may be achievedusing techniques known in the art. In particular, disulphide bonds maybe formed between two appropriately spaced components having freesulfhydryl groups. The bonds may be formed between side chains of aminoacids, non-amino acid components or a combination of the two. Inaddition, the soluble fgl2 protein or peptides of the present inventionmay be converted into pharmaceutical salts by reacting with inorganicacids including hydrochloric acid, sulphuric acid, hydrobromic acid,phosphoric acid, etc., or organic acids including formic acid, aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, succinic acid, malic acid, tartaric acid, citric acid, benzoicacid, salicylic acid, benzenesulphonic acid, and tolunesulphonic acids.

Fragments or portions of the soluble fgl2 protein include fragments orportions of the known fgl2 sequences as well as fragments or portions ofanalogs of soluble fgl2. The inventor has shown that full length mRNAtranscripts of fgl2 (1.3 kb) are made in antigen presenting cells andna{dot over (i)}ve T cells. The inventor has also shown that 5′truncated fgl2 mRNA transcripts are present in Con-A stimulated T cells,indicating that activated T cells secrete truncated fgl2 proteins thatare devoid of a transmembrane domain. The sequence of 3 truncatedsoluble fgl2 molecules is provided in FIGS. 12-14 and SEQ ID NOS:1-6.The sequences of these shorter transcripts reveal that they are notsplice variants but instead are truncations at the 5′ end of the fulllength transcripts, indicating they are the products of alternativetranscriptional start sites. The predicted amino acid sequence of thelongest open reading Frame (indicated by Kozak consensus translationalstart site) of these truncated transcripts corresponds to truncated fgl2proteins that are devoid of the hydrophobic region, which can serve as atransmembrane domain. Thus, these truncated fgl2 proteins can accountfor the soluble fgl2 protein secreted by T cells. Since the hydrophobicregion of fgl2 overlap with its signal peptide sequence, the putativetruncated fgl2 protein will lack the signal peptide necessary for theclassical endoplasmic reticulum-golgi secretion pathway. Therefore, allof the truncated proteins lack the hydrophobic region and the signalpeptide sequence present in the full length fgl2 protein. Accordingly,in one embodiment a truncated soluble fgl2 protein useful in the presentinvention lacks the hydrophobic region of full length fgl2. The inventorhas also shown that exon 2, but not exon 1, of fgl2 is necessary for theimmunosuppressive activity of soluble fgl2. Accordingly, in anotherembodiment, the soluble fgl2 protein useful in the present inventioncomprises the amino acid sequence encoded by exon 2 of the fgl2 gene.

In one embodiment, the soluble fgl2 protein (i) is encoded by a nucleicacid molecule shown in SEQ ID NO:1 (FIG. 12A); (ii) has the amino acidsequence shown in SEQ ID NO:2 (FIG. 12B); or (iii) is an analog of (i)or (ii).

In another embodiment, the soluble fgl2 protein (i) is encoded by anucleic acid molecule shown in SEQ ID NO:3 (FIG. 13A); (ii) has theamino acid sequence shown in SEQ ID NO:4 (FIG. 13B); or (iii) is ananalog of (i) or (ii).

In yet another embodiment, the soluble fgl2 protein (i) is encoded by anucleic acid molecule shown in SEQ ID NO:5 (FIG. 14A); (ii) has theamino acid sequence shown in SEQ ID NO:6 (FIG. 14B); or (iii) is ananalog of (i) or (ii).

The present invention also includes isolated nucleic acid moleculesencoding a soluble fgl2 protein.

The term “nucleic acid sequence” refers to a sequence of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The term also includes modified orsubstituted sequences comprising non-naturally occurring monomers orportions thereof, which function similarly. The nucleic acid sequencesof the present invention may be ribonucleic (RNA) or deoxyribonucleicacids (DNA) and may contain naturally occurring bases including adenine,guanine, cytosine, thymidine and uracil. The sequences may also containmodified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl,2-propyl, and other alkyl adenines, 5-halo uracil, 5-halo cytosine,6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol adenine,8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanines, 8-hydroxyl guanine and other 8-substituted guanines, other azaand deaza uracils, thymidines, cytosines, adenines, or guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

In one embodiment, the nucleic acid molecule encoding soluble fgl2comprises exon 2 of the fgl2 gene.

In a preferred embodiment, the nucleic acid molecule encoding solublefgl2 protein comprises:

(a) a nucleic acid sequence as shown in FIG. 12A (SEQ ID NO:1) or 13A(SEQ ID NO:3) or 14A (SEQ ID NO:5) wherein T can also be U;

(b) a nucleic acid sequence that is complimentary to a nucleic acidsequence of (a);

(c) a nucleic acid sequence that has substantial sequence homology to anucleic acid sequence of (a) or (b);

(d) a nucleic acid sequence that is an analog of a nucleic acid sequenceof (a), (b) or (c); or

(e) a nucleic acid sequence that hybridizes to a nucleic acid sequenceof (a), (b), (c) or (d) under stringent hybridization conditions.

The term “sequence that has substantial sequence homology” means thosenucleic acid sequences which have slight or inconsequential sequencevariations from the sequences in (a) or (b), i.e., the sequencesfunction in substantially the same manner and encode a soluble fgl2protein that is capable of suppressing an immune response. Thevariations may be attributable to local mutations or structuralmodifications. Nucleic acid sequences having substantial homologyinclude nucleic acid sequences having at least 65%, more preferably atleast 85%, and most preferably 90-95% identity with the nucleic acidsequences as shown in FIG. 12A (SEQ ID NO:1) or 13A (SEQ ID NO:3) or 14A(SEQ ID NO:5).

The term “sequence that hybridizes” means a nucleic acid sequence thatcan hybridize to a sequence of (a), (b), (c) or (d) under stringenthybridization conditions. Appropriate “stringent hybridizationconditions” which promote DNA hybridization are known to those skilledin the art, or may be found in Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the followingmay be employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. The stringency may beselected based on the conditions used in the wash step. For example, thesalt concentration in the wash step can be selected from a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be at high stringency conditions, at about 65° C.

The term “a nucleic acid sequence which is an analog” means a nucleicacid sequence which has been modified as compared to the sequence of (a)(b) or (c) wherein the modification does not alter the utility of thesequence as described herein. The modified sequence or analog may haveimproved properties over the sequence shown in (a), (b) or (c). Oneexample of a modification to prepare an analog is to replace one of thenaturally occurring bases (i.e. adenine, guanine, cytosine or thymidine)of the sequence shown in FIG. 12A (SEQ ID NO:1) or 13A (SEQ ID NO:3) or14A (SEQ ID NO:5) with a modified base such as xanthine, hypoxanthine,2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halouracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine,pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyl adenines, 8-hydroxyl adenine and other8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous oroxygen heteroatoms in the phosphate backbone, short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages in the nucleic acid molecule shown inFIG. 12A (SEQ ID NO:1) or 13A (SEQ ID NO:3) or 14A (SEQ ID NO:5). Forexample, the nucleic acid sequences may contain phosphorothioates,phosphotriesters, methyl phosphonates, and phosphorodithioates.

A further example of an analog of a nucleic acid molecule of theinvention is a peptide nucleic acid (PNA) wherein the deoxyribose (orribose) phosphate backbone in the DNA (or RNA), is replace with apolyamide backbone which is similar to that found in peptides (P. E.Nielsen et al, Science 1991, 254, 1497). PNA analogs have been shown tobe resistant to degradation by enzymes and to have extended lives invivo and in vitro. PNAs also bind stronger to a complimentary DNAsequence due to the lack of charge repulsion between the PNA strand theDNA strand. Other nucleic acid analogs may contain nucleotidescontaining polymer backbones, cyclic backbones, or acyclic backbones.For example, the nucleotides may have morpholino backbone structures(U.S. Pat. No. 5,034,506). The analogs may also contain groups such asreporter groups, a group for improving the pharmacokinetic orpharmacodynamic properties of nucleic acid sequence.

II. Therapeutic Methods

In one aspect, the present invention provides a method of suppressing animmune response comprising administering an effective amount of asoluble fgl2 protein or a nucleic acid sequence encoding a soluble fgl2protein to an animal in need of such treatment. The invention alsoincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid sequence encoding a soluble fgl2 protein to suppress animmune response or in the manufacture of a medicament to suppress animmune response.

The term “administering a soluble fgl2 protein” includes both theadministration of a soluble fgl2 protein (as defined above) as well asthe administration of a nucleic acid sequence encoding a soluble fgl2protein. In the latter case, the soluble fgl2 protein is produced invivo in the animal.

Administration of an “effective amount” of the soluble fgl2 protein andnucleic acid of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult, for example suppression of an immune response. The effectiveamount of the soluble fgl2 protein or nucleic acid of the invention mayvary according to factors such as the disease state, age, sex, andweight of the animal. Dosage regima may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The term “suppress” or “reduce” or “inhibit” a function or activity,such as an immune response, means to suppress or reduce the function oractivity when compared to otherwise same conditions in the absence ofsoluble fgl2.

The term “animal” as used herein includes all members of the animalkingdom including humans.

As mentioned previously, the inventor has shown that soluble fgl2inhibits T-cell proliferation, prevents DC maturation and promotes Th2cytokine expression.

In one embodiment, the present invention provides a method of preventingor inhibiting T-cell proliferation comprising administering an effectiveamount of a soluble fgl2 protein or a nucleic acid sequence encoding asoluble fgl2 protein to an animal in need thereof. The inventionincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid sequence encoding a soluble fgl2 protein to prevent orinhibit T-cell proliferation or in the manufacture of a medicament toprevent or inhibit T-cell proliferation.

In another embodiment, the present invention provides a method ofpreventing or inhibiting DC maturation comprising administering aneffective amount of a soluble fgl2 protein or a nucleic acid sequenceencoding a soluble fgl2 protein to an animal in need thereof. Theinvention includes a use of an effective amount of a soluble fgl2protein or a nucleic acid sequence encoding a soluble fgl2 protein toprevent or inhibit DC maturation or in the manufacture of a medicamentto prevent or inhibit DC maturation.

In a further embodiment, the present invention provides a method ofpromoting a Th2 cytokine response comprising administering an effectiveamount of a soluble fgl2 protein or a nucleic acid sequence encoding asoluble fgl2 protein to an animal in need thereof. The inventionincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid sequence encoding a soluble fgl2 protein to promote a Th2cytokine response or in the manufacture of a medicament to promote a Th2cytokine response.

The term “promoting a Th2 cytokine response” means that the levels ofTh2 cytokines is enhanced, increased or induced as compared to thelevels observed in the absence of soluble fgl2. Th2 cytokines include,but are not limited to, IL-4 and IL-10. It is known that promoting a Th2cytokine response is helpful in treating a number of conditionsrequiring immune suppression.

In another aspect of the present invention, there is provided a methodof treating a disease or condition wherein it is desirable to suppressan immune response comprising administering an effective amount of asoluble fgl2 protein or a nucleic acid sequence encoding a soluble fgl2protein to an animal in need of such treatment. The invention alsoincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid sequence encoding a soluble fgl2 protein to treat a diseaseor condition wherein it is desirable to suppress an immune response orin the manufacture of a medicament to suppress an immune response.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminishment of extent of disease, stabilized (i.e. notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder.

The method of the invention may be used to treat any disorder orcondition wherein it is desirable to suppress an immune response, forexample, to induce immune suppression of tolerance to transplantedorgans or tissues, treating graft versus host disease, treatingautoimmune diseases and treating allegies.

As stated hereinabove, the findings that soluble fgl2 inhibited T-cellproliferation, promoted Th2 cytokine expression and prevented DCmaturation suggest a novel usage of soluble fgl2 protein as animmunosuppressive agent which may induce T-cell tolerance and thereforeimprove allograft survival. Accordingly, in an embodiment of the presentinvention, there is provided a method of suppressing an immune responseto a transplanted organ or tissue in a recipient animal comprisingadministering an effective amount of a soluble fgl2 protein or a nucleicacid sequence encoding a soluble fgl2 protein to the recipient animal.The soluble fgl2 can be administered prior to, during or after thetransplantation of the organ or tissue. The invention includes a use ofan effective amount of a soluble fgl2 protein or a nucleic acid sequenceencoding a soluble fgl2 protein to induce immune suppression ortolerance to a transplanted organ or tissue or in the manufacture of amedicament to induce immune suppression or tolerance to a transplantedorgan or tissue.

The term “inducing immune tolerance” means, rendering the immune systemunresponsive to a particular antigen without inducing a prolongedgeneralized immune deficiency. The term “antigen” means a substance thatis capable of inducing an immune response. In the case oftransplantation, immune tolerance means rendering the immune systemunresponsive to the antigens on the transplant. In the case ofautoimmune disease, immune tolerance means rendering the immune systemunresponsive to an auto-antigen that the host is recognizing as foreign,thus causing an autoimmune response. In the case of allergy, immunetolerance means rendering the immune system unresponsive to an allergenthat generally causes an immune response in the host. An alloantigenrefers to an antigen found only in some members of a species, such asblood group antigens. A xenoantigen refers to an antigen that is presentin members of one species but not members of another. Correspondingly,an allograft is a graft between members of the same species and axenograft is a graft between members of a different species.

The recipient can be any member of the animal kingdom including rodents,pigs, cats, dogs, ruminants, non-human primates and preferably humans.The organ or tissue to be transplanted can be from the same species asthe recipient (allograft) or can be from another species (xenograft).The tissues or organs can be any tissue or organ including heart, liver,kidney, lung, pancreas, pancreatic islets, brain tissue, cornea, bone,intestine, skin and haematopoietic cells.

The method of the invention may also be used to prevent graft versushost disease wherein the immune cells in the transplant mount an immuneattack on the recipient's immune system. This can occur when the tissueto be transplanted contains immune cells such as when bone marrow orlymphoid tissue is transplanted when treating leukemias, aplasticanemias and enzyme or immune deficiencies, for example.

Accordingly, in another embodiment, the present invention provides amethod of preventing or inhibiting graft versus host disease in arecipient animal receiving an organ or tissue transplant comprisingadministering an effective amount of a soluble fgl2 protein or a nucleicacid sequence encoding a soluble fgl2 protein to the organ or tissueprior to the transplantation in the recipient animal. The inventionincludes a use of an effective amount of a soluble fgl2 protein or anucleic acid molecule encoding a soluble fgl2 protein to prevent orinhibit graft versus host disease or in the manufacture of a medicamentto prevent or inhibit graft versus host disease.

As stated previously, the method of the present invention may also beused to treat or prevent autoimmune disease. In an autoimmune disease,the immune system of the host fails to recognize a particular antigen as“self” and an immune reaction is mounted against the host's tissuesexpressing the antigen. Normally, the immune system is tolerant to itsown host's tissues and autoimmunity can be thought of as a breakdown inthe immune tolerance system.

Accordingly, in a further embodiment, the present invention provides amethod of preventing or treating an autoimmune disease comprisingadministering an effective amount of a soluble fgl2 protein, or anucleic acid sequence encoding a soluble fgl2 protein to an animalhaving, suspected of having, or susceptible to having an autoimmunedisease. The invention includes a use of an effective amount of asoluble fgl2 protein on a nucleic acid molecule encoding a soluble fgl2protein to prevent or inhibit an autoimmune disease or in themanufacture of a medicament to prevent or inhibit an autoimmune disease.

Autoimmune diseases that may be treated or prevented according to thepresent invention include, but are not limited to, arthritis, type 1insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, meningitis, thromboticthrombocytopenic purpura, Sjögren's syndrome, encephalitis, uveitis,leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever,Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis,primary biliary cirrhosis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Gravesdisease, habitual spontaneous abortions, Reynard's syndrome,glomerulonephritis, dermatomyositis, chronic active hepatitis, celiacdisease, tissue specific autoimmunity, degenerative autoimmunity delayedhypersensitivities, autoimmune complications of AIDS, atrophicgastritis, ankylosing spondylitis and Addison's disease.

One of skill in the art can determine whether or not a particularsoluble fgl2, or fragment thereof, is useful in preventing autoimmunedisease. As mentioned previously, one of skill in the art can readilytest a soluble fgl2 or a soluble fgl2 fragment for its ability tosuppress an immune response using known in vitro assays. In addition thesoluble fgl2 or a soluble fgl2 fragment can also be tested for itsability to prevent autoimmune in an animal model. Further, many otherautoimmune animal models are available, including, but not limited to,experimental allergic encephalomyelitis which is an animal model formultiple sclerosis, animal models of inflammatory bowel disease (inducedby immunization, or developing in cytokine-knockout mice), and models ofautoimmune myocarditis and inflammatory eye disease.

As stated previously, the method of the present invention may also beused to treat or prevent an allergic reaction. In an allergic reaction,the immune system mounts an attack against a generally harmless,innocuous antigen or allergen. Allergies that may be prevented ortreated using the methods of the invention include, but are not limitedto, hay fever, asthma, atopic eczema as well as allergies to poison oakand ivy, house dust mites, bee pollen, nuts, shellfish, penicillin andnumerous others.

Accordingly, in a further embodiment, the present invention provides amethod of preventing or treating an allergy comprising administering aneffective amount of a soluble fgl2 protein or a nucleic acid sequenceencoding a soluble fgl2 protein to an animal having or suspected ofhaving an allergy. The invention includes a use of an effective amountof a soluble fgl2 protein or a nucleic acid molecule encoding a solublefgl2 protein to prevent or treat an allergy.

III. Compositions

The invention also includes pharmaceutical compositions containingsoluble fgl2 proteins or nucleic acids encoding a soluble fgl2 proteinfor use in immune suppression.

Such pharmaceutical compositions can be for intralesional, intravenous,topical, rectal, parenteral, local, inhalant or subcutaneous,intradermal, intramuscular, intrathecal, transperitoneal, oral, andintracerebral use. The composition can be in liquid, solid or semisolidform, for example pills, tablets, creams, gelatin capsules, capsules,suppositories, soft gelatin capsules, gels, membranes, tubelets,solutions or suspensions.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. The pharmaceutical compositions may additionallycontain other agents such as immunosuppressive drugs or antibodies toenhance immune tolerance or immunostimulatory agents to enhance theimmune response.

The present invention provides a pharmaceutical composition for use insuppressing an immune response comprising an effective amount of asoluble fgl2 protein in admixture with a pharmaceutically acceptablediluent or carrier.

In one embodiment, the pharmaceutical composition for use in preventinggraft rejection comprises an effective amount of a soluble fgl2 proteinin admixture with a pharmaceutically acceptable diluent or carrier.

In another embodiment, the pharmaceutical composition for use inpreventing graft rejection comprises an effective amount of a nucleicacid molecule encoding a soluble fgl2 protein in admixture with apharmaceutically acceptable diluent or carrier.

The nucleic acid molecules of the invention encoding a soluble fgl2protein may be used in gene therapy to induce immune tolerance.Recombinant molecules comprising a nucleic acid sequence encoding asoluble fgl2 protein, or fragment thereof, may be directly introducedinto cells or tissues in vivo using delivery vehicles such as retroviralvectors, adenoviral vectors and DNA virus vectors. They may also beintroduced into cells in vivo using physical techniques such asmicroinjection and electroporation or chemical methods such ascoprecipitation and incorporation of DNA into liposomes. Recombinantmolecules may also be delivered in the form of an aerosol or by lavage.The nucleic acid molecules of the invention may also be appliedextracellularly such as by direct injection into cells.

IV. Diagnostic Methods

The finding by the present inventor that soluble fgl2 is involved inimmune suppression allows development of diagnostic assays for detectingdiseases associated with immune suppression.

Accordingly, the present invention provides a method of detecting acondition associated with immune suppression comprising assaying asample for (a) a nucleic acid molecule encoding a soluble fgl2 proteinor a fragment thereof or (b) a soluble fgl2 protein or a fragmentthereof.

To detect nucleic acid molecules encoding soluble fgl2 nucleotide probescan be developed to detect soluble fgl2 or fragments thereof in samples,preferably biological samples such as cells, tissues and bodily fluids.The probes can be useful in detecting the presence of a conditionassociated with soluble fgl2 or monitoring the progress of such acondition. Such conditions include the status of a transplant or anautoimmune disease. Accordingly, the present invention provides a methodfor detecting a nucleic acid molecules encoding a soluble fgl2comprising contacting the sample with a nucleotide probe capable ofhybridizing with the nucleic acid molecule to form a hybridizationproduct, under conditions which permit the formation of thehybridization product, and assaying for the hybridization product.

Example of probes that may be used in the above method include fragmentsof the nucleic acid sequences shown in SEQ ID NOS:1-6. A nucleotideprobe may be labelled with a detectable substance such as a radioactivelabel which provides for an adequate signal and has sufficient half-lifesuch as 32P, 3H, 14C or the like. Other detectable substances which maybe used include antigens that are recognized by a specific labelledantibody, fluorescent compounds, enzymes, antibodies specific for alabelled antigen, and chemiluminescence. An appropriate label may beselected having regard to the rate of hybridization and binding of theprobe to the nucleic acid to be detected and the amount of nucleic acidavailable for hybridization. Labelled probes may be hybridized tonucleic acids on solid supports such as nitrocellulose filters or nylonmembranes as generally described in Sambrook et al, 1989, MolecularCloning, A Laboratory Manual (2nd ed.). The nucleotide probes may beused to detect genes, preferably in human cells, that hybridize to thenucleic acid molecule of the present invention preferably, nucleic acidmolecules which hybridize to the nucleic acid molecule of the inventionunder stringent hybridization conditions as described herein.

Soluble fgl2 protein may be detected in a sample using antibodies thatbind to the protein. Antibodies to soluble fgl2 proteins may be preparedusing techniques known in the art such as those described by Kohler andMilstein, Nature 256, 495 (1975) and in U.S. Pat. Nos. RE 32,011;4,902,614; 4,543,439; and 4,411,993, which are incorporated herein byreference. (See also Monoclonal Antibodies, Hybridomas: A New Dimensionin Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol(eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane(eds.), Cold Spring Harbor Laboratory Press, 1988, which are alsoincorporated herein by reference). Within the context of the presentinvention, antibodies are understood to include monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab′)2) andrecombinantly produced binding partners.

Accordingly, the present invention provides a method for detecting asoluble fgl2 protein comprising contacting the sample with an antibodythat binds to soluble fgl2 which is capable of being detected after itbecomes bound to the soluble fgl2 in the sample.

Antibodies specifically reactive with soluble fgl2, or derivativesthereof, such as enzyme conjugates or labeled derivatives, may be usedto detect soluble fgl2 in various biological materials, for example theymay be used in any known immunoassays which rely on the bindinginteraction between an antigenic determinant of soluble fgl2, and theantibodies. Examples of such assays are radioimmunoassays, enzymeimmunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation,latex agglutination, hemagglutination and histochemical tests. Thus, theantibodies may be used to detect and quantify soluble fgl2 in a samplein order to determine its role in particular cellular events orpathological states, and to diagnose and treat such pathological states.

Accordingly, the invention provides a method for diagnosing a conditionrelated soluble fgl2 expression or activity using an antibody to solublefgl2. In one embodiment, said method comprises obtaining a biologicalsample from a patient (such as a blood or tissue sample) and incubatingsaid sample with an antibody for soluble fgl2, preferably a monoclonalantibody, under conditions that permit formation of a solublefgl2/antibody complex. Said method permits the detection and/ordetermination of the presence and or level of soluble fgl2 in thesample, the presence or particular level of soluble fgl2 beingindicative of a soluble fgl2 related condition. In another embodiment,said antibody is a labelled antibody. In another embodiment, the amountof soluble fgl2 is determined by the amount of complexed soluble fgl2with said soluble fgl2, either directly or indirectly. For instance, ifa particular amount of antibody is used, then the amount of complexed orremaining uncomplexed (or free) antibody can be measured to infer theamount of soluble fgl2 present in the sample.

The following non-limiting examples are illustrative of the presentinvention:

Examples Materials and Methods Mice

Female 6- to 8-wk-old BALB/c (H-2^(d)) and AJ (H-2^(a)) mice werepurchased from Charles River Laboratory (Wilmington, Mass.) and JacksonLaboratory (Bar Harbor, Me.), respectively. All mice were chow-fed andallowed to acclimatize for a week prior experiments.

Reagents

Recombinant mouse (rm) GM-CSF and rm IL-4 were purchased from CedarlaneLaboratories Ltd. (Hornby, ON). LPS (Escherichia coli), human fibrinogenand Con A were purchased from Sigma (St. Louis, Mo.). FITC- orPE-conjugated monoclonal antibodies used to detect cell surfaceexpression of CD80 (16-10A1), CD86 (GL1), CD40 (3/23) and CD11c (HL3),MHC class I (H2-K^(d)), and MHC class II (I-A^(d)) were purchased fromPharMingen (San Diego, Calif.). Anti-Thy-1.2, anti-Ly-2.2 antibodies andrabbit anti-mouse complement were purchased from Cedarlane LaboratoriesLtd. All culture reagents were purchased from Life Technologies(Mississauga, ON) unless otherwise stated.

Production of Purified Soluble fgl2 Protein

Mouse soluble fgl2 protein with a tandem repeat of six histidineresidues followed by an enterokinase cleavage site fused to itsN-terminus was expressed in an Invitrogen Insect Expression System (20).Briefly, a 1.4 kb cDNA encoding mouse fgl2 was amplified using theforward primer 5′-TGCCGCACTGGATCCATGAGGCTTCCTGGT-3′ (SEQ ID NO:7) (withthe methionine start codon underlined) and the reverse primer5′-TTATGGCTTGAAATTCTTGGGC-3′ (SEQ ID NO:8) (nt 1283 to 1302 relative tothe ATG start codon). Amplification was performed for 25 cycles with 2min at 96° C., 2 min at 55° C. and 3 min at 72° C. The PCR product wascloned into the EcoR1 and BamH1 sites of the vector pBlueBacHis2A(Invitrogen).

Putative recombinant viruses were generated according to the Invitrogenprotocol and screened for the presence of fgl2 by PCR followed by threerounds of viral plaque purification. The sequence of the recombinantbaculovirus containing mouse fgl2 cDNA was confirmed by an automated DNAsequencer (Applied Biosystems, model 377, Perkin-Elmer).

Monolayers of High 5 insect cells were infected with the recombinantbaculovirus for the expression of mouse fgl2 protein. Seventy-two hourslater, the infected cells were harvested by centrifugation and lysed in6 M guanidinium hydrochloride, 20 mM sodium phosphate and 500 mM NaCl.The soluble material was mixed with 50% slurry of ProBond Nickel-NTA(Ni-NTA) resin (Invitrogen) for 1 h at 4° C. After washings, bound fgl2protein was eluted with 8 M urea, 20 mM sodium phosphate, pH 5.3 with150 mM NaCl. The pH of the eluted protein was adjusted to pH 7.2immediately upon elution, and the protein was renatured by dialyzingagainst urea-saline buffers (150 mM NaCl, pH 7.2) with successivedecreases in urea concentrations (6M, 4M, 2M, 1M) and finally againstTris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.2). The dialyzedmaterial was concentrated and soluble fgl2 protein was collected bycentrifugation at 14,000 rpm for 10 min to remove insolubleparticulates. Protein concentrations were determined by a modified Lowrymethod, Bicinchoninic Acid (BCA) assay (Pierce; Brockville, ON).

Homogeneity of purified soluble fgl2 protein was evaluated by SDS-PAGEand confirmed by Western blot probed with anti-fgl2 antibody aspreviously described (16). Proteins were stained directly usingCoomassie brilliant blue or were transferred to nitrocellulose, andprobed using polyclonal rabbit anti-mouse fgl2 IgG as the primary Ab.The secondary Ab utilized for immunoblotting was affinity-purifieddonkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham,Buckinghamshire, UK) and the blot was visualized with animmunochemiluminescent kit (Amersham).

Biotinylation of Soluble fgl2 and Bovine Serum Albumin (BSA)

A 1mg/ml solution of purified soluble fgl2 and Ig-free BSA (Sigma) wereincubated with a 1:10 molar reaction mixture ofD-Biotinyl-ε-aminocaproic acid-N-hydroxy-succinimide ester(Biotin-7-NHS) (Roche Diagnostics, Laval, QC) for 1 h at roomtemperature with gentle mixing. The reaction was then applied to aSephadex G-25 column (Roche Diagnostics), washed with 1.5 ml of PBS,then the biotinylated-soluble fgl2 was eluted with 3.5 ml of PBS. Theeluate was collected and the protein concentration was determined by amodified Lowry method, Bicinchoninic Acid (BCA) assay. The labeledproteins were utilized in binding assays as described below.

Preparation of Cells

Spleen, lymph node and bone marrow cell suspensions were preparedaseptically. Spleen mononuclear cells were isolated by standardLympholyte-M density gradient (Cedarlane). All cell suspensions wereresuspended in complete medium (α-MEM, supplemented with 10% FBS, 50 μMβ-mercaptoethanol, 1 mM L-glutamine, 100 U/ml penicillin and 100 mg/mlstreptomycin).

Bone marrow-derived DC were prepared as described elsewhere (21).Briefly, bone marrow cells were removed from femurs and tibias of BALB/c(H-2^(d)) mice and filtered through nylon mesh. Cells were incubatedwith anti-Thy-1.2 on ice for 45 min and then treated with rabbitanti-mouse complement for an h at 37° C. The cells were washed andcultured in 100 mm tissue culture dishes in complete medium supplementedwith 10% FBS (Flow Laboratories, Mississauga, Canada) at a concentrationof 1×10⁶ cells/ml with recombinant mouse (rm) GM-CSF (800 U/ml) and rmIL-4 (500 U/ml). On day 2 and 4, nonadherent granulocytes were discardedand fresh rm GM-CSF and rm IL-4 were added at 36-h intervals. ImmatureDC were collected on day 7 and LPS (1 μg/ml) was added to the culturefor 24 h to allow for maturation.

Assays for T Cell Proliferation

All assays were performed in 96-well U-bottom microtiter plates (FalconPlastics, N.J.) in a humidified atmosphere with 5% CO₂ at 37° C.

1. Alloantigen Stimulation

For alloantigen stimulation, Balb/c splenic mononuclear cells (4×10⁵cells/100 μl) were stimulated with irradiated (3000 rad) A/J splenicmononuclear cells (4×10⁵ cells/100 μl) with or without soluble fgl2protein (ranging from 1 μg/ml to 1 ng/ml) added to the culture.

2. Response to Concanaviin A (Con A)

Purified T cells (2×10⁵ cells/200 μl) were stimulated with Con A (5μg/ml; Sigma) in the presence or absence of soluble fgl2 protein(ranging from 1 μg/ml to 1 ng/ml). After 3 d, cells were pulsed with[³H]thymidine (1 μCi/well) (Amersham Biosciences; Piscataway, N.J.) for18 h prior to harvesting and determining the incorporated radioactivity.

3. Response to Anti-CD3 and Anti-CD28

Purified T cells (2×10⁵ cells/200 μl) were stimulated with immobilizedanti-CD3 mAb (1 μg/ml) and soluble anti-CD28 mAb (20 ng/ml) in thepresence or absence of soluble fgl2 protein (ranging from 1 μg/ml to 1ng/ml). After 3 d, cells were pulsed with [³H]thymidine (1 μCi/well)(Amersham Biosciences; Piscataway, N.J.) for 18 h prior to harvestingand determining the incorporated radioactivity.

H5 supernatants, wild type baculovirus infected H5 supernatants, humanfibrinogen (1 μg/ml) and bovine serum albumin (1 μg/ml) were added tothe culture in parallel with soluble fgl2 protein as controls aspreviously described (20).

In some experiments, a monoclonal Ab against the “domain 2”(FRED-containing C terminal region) of fgl2 (1 μg) was added at thebeginning of culture along with soluble fgl2 protein. An isotype controlAb was added for comparison.

Allogeneic Mixed Leukocyte Reaction

DC (1×10⁴) obtained from the bone marrow of A/J mice were firststimulated with LPS as described above, irradiated and then were mixedwith responder BALB/c lymph node T cells (2×10⁵) in 96-well U-bottommicrotiter plates for 48 h. Purified soluble fgl2 protein (1 μg/ml or 1ng/ml) was added at the beginning of cultures. Proliferation wasmeasured by pulsing after 2 d of culture with [³H]thymidine (1 μCi/well)for 18 h as described above. In cultures used to assess cytokineproduction, supernatants were pooled from triplicate wells at 40 h.Levels of IL-2, IL-4, IFN-γ, and IL-10 were assayed using ELISA kits(Pierce) according to the manufacturer's instructions. Where CTLinduction was assayed, cultures were allowed to continue for 5 days (inthe presence/absence of soluble fgl2), before cells were harvested.These effector cells were assayed in standard 4 hr ⁵¹Cr-release assaysat various effector:target ratios with ⁵¹Cr-labeled 72 hr-Con Aactivated A/J blast target cells, as described elsewhere (21). Data wereexpressed as a percent specific lysis at 50:1 effector:target.

The effect of soluble fgl2 on the LPS-induced maturation of BM-derivedDC was examined by adding soluble fgl2 protein (1 μg/ml) to DC culturesduring LPS-induced maturation. The treated DC cultures were then washedand examined for their ability to stimulate alloreactive T cellproliferation as described above. The expression of surface moleculesincluding CD40, CD80, CD86, CD11c, MHC class I and class II moleculeswere measured by flow cytometric analysis. In other experiments, lymphnode T cells were exposed to soluble fgl2 protein (1 μg/ml) for 12 h,washed then cultured with allogeneic DC. Proliferation was measured bypulsing after 2 days of culture with [³H]thymidine (1 μCi/well) for 18 has described above.

One-Way Xenogeneic Mixed Lymphocyte Reaction (MLR)

Female Wistar rats (150 g, Charles River, Wilmington, Mass.) were usedas xenogeneic skingraft donors that were engrafted onto Balb/c mice for13 days prior to being sacrificed. Then, splenic mononuclear cells(1×10⁶ cells/100 μl) from skingraft recipients were harvested asresponder cells and restimulated in vitro with irradiated (3000 rad)Wistar splenic mononuclear cells (1×10⁶ cells/100 μl) in 96-wellU-bottom microtiter plates with or without purified soluble fgl2 protein(from 0.1 μg/ml to 1 μg/ml). The culture was incubated at 5% CO₂, and37° C. for 3 days and then the cells were pulsed with of [³H] thymidine(1 μCi/well) (Amersham) for 18 hr before harvesting. Cell proliferationwas quantified by [³H] thymidine incorporation using a TopCountβ-counter (Canberra-Packard Canada Ltd., Mississauga, ON).

Flow Cytometric Analysis

To examine the binding of biotinylated-soluble fgl2 to peripheral Tcells or BM-derived DC, cells were washed twice with PBS, blocked with10% v/v normal mouse serum for 5 min at room temperature and thenincubated with biotinylated-soluble fgl2 protein in PBS at 4° C. for 30min. Cells were washed extensively, stained with SA-PE (Pharmingen) at4° C. for 30 min then analyzed on COULTER Epics-XL-MCL flow cytometer(Beckman Coulter, Fla.) using XL software. Binding ofbiotinylated-soluble fgl2 on T cells and DC was analyzed on CD3- andCD11c-positive cells, respectively. Cells incubated withbiotinylated-BSA then SA-PE were used as negative controls.

For characterization of the prepared DC population, 2×10⁵ cells werefirst blocked with 10% v/v normal mouse serum for 5 min at roomtemperature and thereafter stained with the corresponding FITC- orPE-conjugated mAb in PBS with 1% BSA at 4° C. for 30 min. Cells stainedwith the appropriate isotype-matched Ig were used as negative controls.Cells were analyzed on COULTER Epics-XL-MCL flow cytometer forexpression of various DC markers.

To assess cell cycle and apoptosis, cells treated with soluble fgl2protein for 12 h were washed in cold PBS, resuspended in lysis buffer(0.1% sodium citrate/Triton X-100) containing 100 units/ml RNase A(Sigma) and stained with propidium iodide (PI) (1 mg/ml PBS) in the darkfor 20 min at room temperature. The cells were then washed twice andapproximately 10,000 data events per sample were analyzed. Gates wereset, by using the untreated sample, to differentiate between G₀/G₁(left-hand peak), S-phase (intermediate) and G₂/M (right-hand peak).Apoptotic cells appeared to the left of the G0/G1 phase.

Immunofluorescence Microscopy

The effect of soluble fgl2 on LPS-induced NF-κB translocation wasexamined as previously described (22). Immature DC were harvested on day7 and were allowed to adhere to autoclaved glass coverslips for 6 h at37° C., 5% CO₂, incubated in complete medium supplemented 10% FBS,GM-CSF and recombinant mouse IL-4 as described above. To examine theeffects that soluble fgl2 had on LPS-induced NF-κB translocation,soluble fgl2 protein (1 μg/ml) was added to the DC cultures during LPS(1 μg/ml) stimulation. NF-κB translocation was examined at 5, 15, 30,60, 120, 240 min. Cells were fixed for 30 min in PBS supplemented with2% paraformaldehyde. The coverslips were washed three times with PBS for10 min each, permeabilized with 0.2% Triton X-100 in PBS for 5 min, andthen blocked with 5% BSA in PBS for 30 min at room temperature. Thesamples were stained with a goat anti-p65 polyclonal antibody (1:50dilution in PBS) (Molecular Probes Inc. Eugene, Oreg.) for 1 hr at roomtemperature, washed three times with PBS for 5 min each, and incubatedwith fluorescently labeled Alexa 555 donkey anti-goat IgG secondaryantibody (1:400 dilution in PBS) (Molecular Probes Inc., Eugene, Oreg.)for 1 hr at room temperature. The coverslips were washed three timeswith PBS for 5 min each and mounted on glass slides using mountingsolution (DAKO from Dakocytomation, Carpinteria, Calif.). Nuclei werecounterstained with DAPI (4′,6′-diamidino-2-phenylindole) chromosomalstaining (Molecular Probes Inc, Eugene, Oreg.). The staining wasvisualized using a Nikon TE200 fluorescence microscope (X100 objective)coupled to Orca 100 camera driven by Simple PCl software as previouslydescribed (22).

Statistical Analysis

The results were calculated as means±standard error of the mean (SEM).For statistical comparison, the means were compared using the analysisof variance by Student's t-test using the software Statistix 7(Analytical Software). A “p” value≦0.05 was considered statisticallysignificant.

Example 1

Generation of Soluble fgl2 Protein in a Baculovirus Expression System

To characterize the immunomodulatory property of soluble fgl2, fgl2protein was generated using a baculovirus expression system and purifiedas described in the Methods. SDS-PAGE followed by Coomassie bluestaining of the purified soluble fgl2 protein showed a dominant band at65-kDa, comparable to the size of the fgl2 protein previously reported(15, 16) (FIG. 1A). Purified soluble fgl2 was confirmed by Westernblotting using polyclonal rabbit anti-mouse fgl2 IgG (FIG. 1B). Thepurified soluble fgl2 protein was analyzed for its ability to induceclotting and no coagulation activity was detected (data not shown).

Soluble fgl2 Binding to T Cells and DC

The binding of purified soluble fgl2 to T cells and DC was examinedusing flow cytometry analysis. FIG. 2 shows that biotinylated-solublefgl2 bound to both T cells and DC. The specific binding ofbiotinylated-soluble fgl2 to both cells was inhibited bynon-biotinylated soluble fgl2 but not by fibrinogen (data not shown).

Soluble fgl2 Inhibited T Cell Proliferation Stimulated by VariousStimuli

To examine the consequence of the binding of soluble fgl2 to T cells,purified soluble fgl2 protein was initially assessed for its capacity toinhibit T cell proliferation. FIG. 3 shows that soluble fgl2 proteininhibited allogeneic T cell activation in a dose-dependent manner. Atthe highest concentration of soluble fgl2 (1 μg/ml) used in thecultures, 61±11% inhibition of T cell proliferation was observed.Purified soluble fgl2 protein similarly inhibited T cell proliferationinduced by immobilized anti-CD3 mAb with soluble anti-CD28 mAb and byCon A (FIGS. 3 b, c) in a dose dependent fashion. H5 supernatants, wildtype baculovirus infected H5 supernatants, human fibrinogen (1 μg/ml)(FIG. 3) nor bovine serum albumin (1 μg/ml) (data not shown) had anyeffect on T cell proliferation stimulated by alloantigen, anti-CD3/CD28or Con A.

Soluble fgl2 Inhibited Allogeneic Response at Early Time Points and theEffect Could be Neutralized by Monoclonal Antibody

To further explore the suppressive effect of soluble fgl2 onalloreactive T cell proliferation, soluble fgl2 protein was added toallogeneic cultures at different time points (i.e. day 0, 1, 2 or 3) andmixed by pipetting to ensure proper distribution of protein to thecultures. Cell proliferation was measured as previously described.Cultures without the addition of soluble fgl2 were also mixed bypipetting at corresponding time points as controls. FIG. 4 a shows thatsoluble fgl2 exhibited maximal inhibitory effect (61±11% inhibition)when it was added at the initiation of allogeneic reactions (day 0).Less inhibitory effects were observed when soluble fgl2 was added on day1 (39±15% inhibition) with loss of inhibition when addition of solublefgl2 was delayed until day 2.

The ability of a monoclonal antibody (mAb) against the “domain 2”(FRED-containing C terminal region) of mouse fgl2 to neutralize theinhibitory effect of soluble fgl2 on alloreactive T cell proliferationwas next examined. As shown in FIG. 4 b, a mouse mAb (1 μg/ml) abrogatedthe ability of soluble fgl2 to suppress alloreactive T cellproliferation, suggesting that the effect of soluble fgl2 protein wasspecific and could be prevented by this Ab. In contrast, a rabbitpolyclonal Ab against the “domain 1” of fgl2 which neutralizes thecoagulation activity of fgl2 failed to inhibit the immunosuppressiveactivity of soluble fgl2 protein.

Soluble fgl2 Promoted a Th2 Cytokine Profile in Allogeneic Responses

To characterize further the effect of soluble fgl2 on allogeneicresponses, the inventor cultured T cells with irradiated allogeneicBM-derived LPS-induced mature DC in the presence of soluble fgl2protein. A similar dose-specific soluble fgl2 suppressive effect onalloreactive T cell proliferation was observed to that seen in FIG. 3.FIG. 5 a shows that 1 μg/ml of soluble fgl2 protein resulted in amaximal 68±14% inhibition of T cell proliferation. Supernatantscollected from these cultures (1 μg/ml of soluble fgl2) showed decreasedlevels of IL-2 and IFN-γ, no effect on levels of IL-12 and increasedlevels of IL-4 and IL-10 productions in comparison to supernatants fromsoluble fgl2-untreated allogeneic cultures (FIGS. 5B & C). Thepossibility that this alteration in cytokine response was due to directtoxicity was further examined. Soluble fgl2 did not cause non specificchanges in cell survival of stimulated or unstimulated cells asdiscussed further below. In addition, the inventor did not observe anyinhibition of CTL induction in the presence of soluble fgl2. Thuspercent lysis at 5 days in control cultures (no fgl2) at 50:1effector:target was (30±5%), whereas in fgl2-treated cultures lysis was31±3% (p=0.74). The addition of soluble fgl2 protein to the allogeneiccultures at a concentration of 1 ng/ml had no inhibitory effect on Tcell proliferation, and resulted in a promotion of Th1 cytokinesexpression, similar to that observed in soluble fgl2 untreated cultures(FIGS. 5B & C).

Soluble fgl2 Did Not Suppress T Cell Proliferation Via Induction ofApoptosis

Others have reported that certain immunosuppressive agents suppress Tcell proliferation by inducing T cell apoptosis (23). Therefore, T cellviability was examined by both trypan blue dye exclusion and PI stainingof lymphocyte nuclei after a 12 h-exposure to soluble fgl2 protein. Atall concentrations of soluble fgl2 tested in this study, no significantdifferences in cell number and amount of apoptotic cells were detectedbetween the soluble fgl2-treated T cells and untreated T cells (data notshown), suggesting that the inhibitory effects of soluble fgl2 were notan outcome of a nonspecific or cytotoxic effect.

Soluble fgl2 Led to Reduced Expression of CD80^(hi) and MHC Class IIIMolecules by BM-Derived DC

The inventor next examined whether soluble fgl2 had the ability toimpair the maturation of BM-derived DC. To test this, immature DC weregenerated by culturing BM cells with GM-CSF and IL-4 for 7 d. Followingthe addition of LPS, in the presence or absence of soluble fgl2 (1μg/ml), the phenotype of these DC were examined by staining of cellswith various mAbs followed by flow cytometry analysis. As shown in FIG.6 a, CD11c⁺ cells composed the majority of both soluble fgl2-treated andnon-treated cells, suggesting that the addition of soluble fgl2 did notreduce the number or viability of LPS-treated DC. Furthermore, theexpression of MHC class I and CD86 was not altered by soluble fgl2(FIGS. 6 b and 6 c). A minor change in CD40 expression was observed(FIG. 6 f). However, incubation of DC with soluble fgl2 duringLPS-induced maturation significantly reduced expression of both,CD80^(hi) (FIG. 6 d) MHC class II^(hi) (FIG. 6 e) and CD80^(hi) (FIG. 6d) expression on DC. These findings suggest that soluble fgl2 inhibitsLPS-induced DC maturation.

Addition of Soluble fgl2 During DC Maturation Abolished Their Ability toInduce Allogeneic Responses

To further examine the effect of soluble fgl2 protein on DC maturation,the inventor determined the ability of soluble fgl2-treated DC tostimulate allogeneic responses. FIG. 7 shows that DC treated withsoluble fgl2 (1 μg/ml) during the LPS-induced maturation had an impairedability to 5 stimulate naive allogeneic T cell proliferation, incomparison to soluble fgl2-untreated DC. When naive T cells werepretreated with soluble fgl2 protein (1 μg/ml) for 12 h, washed, thencultured with allogeneic LPS-induced mature DC, no inhibitory effect onT cell proliferation was observed. This was compared to the levels ofproliferation observed in cultures containing untreated control T cellsstimulated with allogeneic LPS-induced mature DC. Maximal abrogation onalloreactive T cell proliferation was resulted when na{dot over (i)}ve Tcells were stimulated with soluble fgl2-preexposed DC in the presence ofsoluble fgl2 protein (1 μg/ml).

To examine whether soluble fgl2 prevents the maturation of BM-derived DCthrough the NF-κB pathway, BM-derived DC were stimulated with LPS (1μg/ml) following 7 days of incubation in GM-CSF and recombinant mouseIL-4 in the presence or absence of soluble fgl2 protein (1 μg/ml) whichwas added at the same time of LPS exposure. The dendritic cells wereexamined at 5, 15, 30, 60, 120, and 240 min for NF-κB translocation byIF microscopy. To clearly determine if there was nuclear translocation,dual staining with a primary antibody to the p65 subunit of NF-κB andDAPI nuclear staining was used. Translocation of NF-κB occurred at alltimes examined, but was maximal after 1 h of LPS stimulation. NF-κBtrans location was significantly reduced by the presence of soluble fgl2protein at all time points examined (FIG. 8).

Discussion

The inventor has had a long interest in defining the regulation ofinduction and mechanism(s) of action of fgl2/fibroleukin, a novelprotein that is expressed by both reticuloendothelial cells (macrophagesand endothelial cells) and T cells. Fgl2 is a 432-amino acid proteinthat shares homology to the β and γ chains of fibrinogen with a FRED atthe carboxyl-terminus (amino acids 202-432). When fgl2 is expressed as amembrane-associated protein in activated macrophages and endothelialcells, it exhibits a coagulation activity capable of directly cleavingprothrombin to thrombin. The membrane associated-fgl2 prothrombinasewith the ability to directly generate thrombin plays an important rolein innate immunity.

A protein belonging to the fibrinogen-like superfamily has been shown toexhibit immunomodulatory property. Tenascin, which shares a 40% homologyto the FRED region of fgl2, blocks T cell activation induced by asoluble antigen, alloantigens, or ConA. The mechanism by which tenascinblocks T cell activation remains undefined. Recently, a soluble form offgl2/fibroleukin (soluble fgl2) has been described, and of particularinterest to the current study is the discovery that T cells are known toexpress soluble fgl2. Nevertheless, the function(s) of soluble fgl2remains unexplored.

The inventor examined the role of soluble fgl2 in regulating thefunction of APC, in particular, DC. BM-derived DC were prepared and theeffect of soluble fgl2 on LPS-induced maturation was examined. Theinventor found that soluble fgl2 prevented the maturation of BM-derivedDC by inhibiting the expression of CD80^(hi), and MHC class II^(hi)molecules, while having no significant effects on MHC class I, CD11c andCD86 expression. These data are consistent with the observation thatsoluble fgl2-treated DC had a markedly reduced capacity to stimulate Tcell proliferation in an allogeneic MLR and inhibit a Th1 cytokineresponse. Interestingly, further abrogation on alloreactive T cellproliferation was achieved when na{dot over (i)}ve T cells werestimulated with soluble fgl2-preexposed DC in the presence of solublefgl2 protein (1 μg/ml), suggesting that soluble fgl2 exerts animmunosuppressive effect on T cells in addition to its effect on DCmaturation.

The inventor has, in addition, explored evidence for a more directeffect of soluble fgl2 on T cells by examining the effect of solublefgl2 on T cells by exploring its effect on T cell proliferationstimulated under different stimuli. In this study, the inventor showedthat soluble fgl2 inhibited T cell proliferation induced by alloantigen,anti-CD3/CD28 or Con A. Although fgl2 shares a 36% homology to the β andγ chains of fibrinogen within the FRED, fibrinogen did not exhibit animmunosuppressive effect on T cell proliferation. This suggests thespecificity of the immunosuppressive effect of soluble fgl2 on T cellproliferation.

The hypothesis that soluble fgl2 has a direct influence on T cells issupported by the inventor's findings that soluble fgl2 suppresses T cellproliferation induced by anti-CD3/CD28 mAbs and Con A. Soluble fgl2 mayalso act directly on APC to inhibit T cell proliferation in MLC.

The immunosuppressive effects of soluble fgl2 on alloreactive T cellproliferation was neutralized by a mAb having no inhibitory effect onthe coagulation activity of fgl2, a function which is known to reside in“domain 1” of the molecule, a region distinct from “domain 2” which isthe FRED-containing C terminal region. Similarly, a polyclonal Ab whichpossesses the ability to neutralize the fgl2 prothrombinase activity andwhich interacts with “domain 1” of the fgl2 molecule had no inhibitoryeffect on the immunosuppressive activity of soluble fgl2. Takentogether, the inventor postulates that distinct domains of fgl2 areresponsible for the prothrombinase and immunomodulatory activities ofthe molecule.

DC themselves are professional APCs which exhibit an ability tostimulate both naive and memory T lymphocytes following their maturation(24, 25). The DC maturation process involves increased expression ofsurface MHC class II and costimulatory molecules and occurs in vivo asDC pass from the periphery to T cell areas of secondary lymphoid tissue.BM-derived DC deficient in costimulatory molecules can induce T cells toundergo a state of hyporesponsiveness, leading to prolongation of isletand cardiac allograft survival (25, 26) and inhibition of autoimmunedisease progression in a variety of animal models (27).

The mechanism(s) by which soluble fgl2 alters the expression of CD80 andMHC class II was examined in the present studies. Nuclear translocationof members of the NF-κB family, particularly RelB, have been shown to berequired for myeloid DC maturation (27, 31, 32). By immunflourescencemicroscopy, it was shown that soluble fgl2 markedly inhibits NF-κBtranslocation which may account for lack of maturation of DC asindicated by lack of expression of CD80^(hi) and MHC Class II. The factthat not all DC were inhibited by soluble fgl2 may reflect dosagerequirements as well as the fact that the population of DC are nothomogeneous.

In the present studies, soluble fgl2 was shown to promote a Th2-cytokineprofile (IL-4 and IL-10) during the initiation of the allogeneicresponse. Cytokines produced by Th2 cells have been shown to exhibitanti-inflammatory activities by regulating the development and activityof Th1 cells, which are in general associated with the development ofautoimmunity, delayed-type hypersensitivity (DTH) and cell-mediatedimmune responses (33-35). Both IL-4 and IL-10 have been shown toantagonize development of Th1 cells, likely through decreasingexpression/function of the cytokine IL-12, while promoting thedifferentiation of Th2 cells. In human and animal studies, polarizationtowards type-2 cytokine production has been associated with improvedsurvival of allogeneic transplants (21, 35). Whether soluble fgl2 wouldaffect graft survival in transplantation remains to be examined. Note inthis report that soluble fgl2 had no effect on CTL activity in an allomixed lymphocyte culture (MLC).

The actual mechanism(s) by which soluble fgl2 promotes a Th2 cytokinedifferentiation is not known. However, it is known that thedifferentiation of na{dot over (i)}ve CD4+T cells into differentpopulations of cytokine-secreting effector cells is influenced not onlyby the cytokine milieu in which differentiation takes place, but by avariety of accessory molecule interactions. The interaction of CD28 withCD86 (36, 37), CD4 with MHC class II (38, 39) and Ox-40 with Ox-40ligand (40) have all been suggested to promote Th2 differentiation atthe expense of Th1 differentiation, whereas CD28 interaction with CD80on antigen presenting cells such as DC have been proposed to produce aTh1 response. Thus, preservation of CD86 and loss of CD80 and MHC ClassII may explain the preferential bias towards Th2 cytokine productionobserved.

In summary, the inventor has reported that while membrane-bound fgl2acts as a prothrombinase, soluble fgl2 is an immunomodulatory proteinwhich has the ability to modulate T cell responses, and perhaps moreimportantly, alter DC maturation to favor production of tolerogenic DC.Currently, the use of non-specific immunosuppressive drugs to treattransplant rejection and autoimmune diseases is fraught withcomplications caused by drug toxicity and other adverse(immunologically) non-specific side effects. The inhibition of CD80interaction with CD28 has been shown to have significantimmunosuppressive effects including (but not limited to) the reductionof specific Ab production; prolongation of the survival of organtransplants; and the inhibition of autoimmune diabetes and lupus. Thusthe direct immunosuppressive activity of soluble fgl2 on T cells and itsability to prevent the expression of costimulatory molecules onLPS-stimulated DC would allow potential strategy in treating autoimmunedisorders and transplant rejection.

Here the inventor shows that soluble fgl2 has a potent immunomodulatoryfunction. The findings that soluble fgl2 inhibited T-cell proliferation,promoted Th2 cytokines expression and prevented DC maturation suggest anovel usage of soluble fgl2 protein as an immunosuppressive agent whichmay impair DC maturation. This has broad implications in the treatmentof allograft rejection and other immune-regulated diseases.

Example 2 Immunomodulatory Effects of Soluble Fgl2

Fibrinogen-related proteins have been shown to have immuoregulatoryactivities. It is believed that fgl2 also exerts immunoregulatoryfunctions due to the presence of a FRED region near its C-terminal. Totest this, the immunoregulatory property of fgl2 was tested in a one-wayxenogeneic mixed lymphocyte reaction (see Methods). A rat (Wistar) tomouse (Balb/c) skingraft transplant was performed and the mice wereprimed with the skingraft for 13 days. Responder T cells were harvestedfrom the lymph nodes of the skingraft recipient and stimulated withmitomycin C inactivated splenic cells of the Wistar rat origin in vitroin the presence of purified fgl2 protein. FIG. 9 shows that fgl2 proteininhibited xenogeneic T cell proliferation in a dose-dependent manner. Atthe highest concentration of fgl2 protein (μg/ml) used in theexperiment, 30±8.4% inhibition of T cell proliferation was observed.

Example 3 Identification of Putative Fgl2 Molecular Variants

It has been shown that fgl2 is a secreted protein in T cells but is alsobelieved that fgl2 is a type II transmembrane protein in macrophages dueto the presence of a hydrophobic region near the N-terminal and therequirement of phospholipids to function as a prothrombinase. In orderto determine whether structural protein variants existed to account forboth a secreted and a transmembrane form of fgl2 protein, various celltypes of the immune system were examined for putative fgl2 mRNAmolecular variants that could generate structurally distinct proteins.5′ Rapid Amplification of cDNA Ends (5′RACE) analysis of murine fgl2gene expression was performed on mRNAs isolated from naive T cells, ConA-stimulated T cells (5 μg/ml Con A stimulation for 3 days), naive Bcells, LPS-stimulated B cells (10 μg/ml LPS for 3 days), immature andmature dendritic cells, naive macrophages, and IFN-γ-stimulatedmacrophages (100 U/ml IFN-γ for 24 hrs). FIG. 10 shows full length 1.3kb mRNA fgl2 transcripts were expressed in na{dot over (i)}ve T cells aswell as the antigen presenting cells: LPS-stimulated B cells, immatureand mature dendritic cells, naive macrophages, and IFN-γ-stimulatedmacrophages. In contrast, no fgl2 mRNA transcript was present in na{dotover (i)}ve B cells and Con A-stimulated T cells expressed three shortermRNA fgl2 transcripts corresponding to molecular sizes, 1.2 kb, 1.0 kband 0.8 kb.

Example 4 Characterization of Fgl2 Molecular Variants in ConcanavalinA-stimulated T Cells

The three shorter fgl2 mRNA transcripts seen in Con A-stimulated T cellscan be the result of alternative splicing at the exon-intron junction,or alternative transcriptional start sites at the 5′ end of the gene. Inorder to characterize these fgl2 mRNA variants, the 5′RACE products werepurified from the agarose gel, subcloned into the vector, TOPO pCR2.1and the inserts of the resulting plasmids were subsequently sequencedusing primers specific for TOPO pCR2.1. The sequences of the 1.3 kb bandobserved in the antigen presenting cells and na{dot over (i)}ve T cells(FIG. 10) corresponded to the full length fgl2 mRNA that was previouslydescribed as necessary to express the fgl2 protein with theprothrombinase activity (FIG. 11) (42). The sequences of the threeshorter fgl2 mRNA transcripts (1.2 kb, 1.0 kb and 0.8 kb shown in FIGS.12-15) revealed the partial deletions of the transcripts were not due toalternative splicing at the exon-intron junction but rather the resultof 5′ end truncation of the mRNAs indicating they were the products ofalternative transcriptional start sites (FIG. 11). The longest openreading frames of the 1.2 kb, 1.0 kb and 0.8 kb fragments as determinedby the first methionine codon with a Kozak consensus sequence wouldbegin translation at the ATG nucleotide position +217, +475, and +577,respectively (with the reference position of the first methionine ATGcodon leading to the translation of the full length fgl2 protein as +1,FIG. 11). These open reading frames are also in frame with the one thatgenerates a full length fgl2 protein with the prothrombinase activity.All three of the predicted amino acid sequence of the truncated fgl2proteins lack the hydrophobic region and the signal peptide sequencepresent in the full length fgl2 protein.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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1-20. (canceled)
 21. A method of suppressing an immune responsecomprising administering an effective amount of a soluble fgl2 proteinto an animal in need of such treatment.
 22. The method according toclaim 21 to suppress an immune response to a transplanted organ ortissue.
 23. The method according to claim 21 to treat a disease orcondition wherein it is desirable to suppress an immune response. 24.The method according to claim 23 to treat an autoimmune disease.
 25. Amethod according to claim 21 to inhibit T cell proliferation.
 26. Amethod according to claim 21 to inhibit dendritic cell maturation.
 27. Amethod according to claim 21 to promote a Th2 cytokine response.
 28. Themethod according to claim 21 wherein the soluble fgl2 protein is a humansoluble fgl2 protein or a fragment thereof.
 29. The method according toclaim 21 wherein the soluble fgl2 comprises domain 2 from the fulllength fgl2 protein.
 30. The method according to claim 21 wherein thesoluble fgl2 protein (i) is encoded by the nucleic acid molecule shownin SEQ ID NO:1 (FIG. 12A); (ii) has the amino acid sequence shown in SEQID NO:2 (FIG. 12B); or (iii) that is an analog of (i) or (ii).
 31. Themethod according to claim 21 wherein the soluble fgl2 protein (i) isencoded by the nucleic acid molecule shown in SEQ ID NO:3 (FIG. 13A);(ii) has the amino acid sequence shown in SEQ ID NO:4 (FIG. 13B); or(iii) that is an analog of (i) or (ii).
 32. The method according toclaim 21 wherein the soluble fgl2 protein (i) is encoded by the nucleicacid molecule shown in SEQ ID NO:5 (FIG. 14A); (ii) has the amino acidsequence shown in SEQ ID NO:6 (FIG. 14B); or (iii) that is an analog of(i) or (ii).
 33. A soluble fgl2 protein (i) encoded by the nucleic acidmolecule shown in SEQ ID NO:1 (FIG. 12A); (ii) having the amino acidsequence shown in SEQ ID NO:2 (FIG. 12B); or (iii) that is an analog of(i) or (ii).
 34. A soluble fgl2 protein (i) encoded by the nucleic acidmolecule shown in SEQ ID NO:3 (FIG. 13A); (ii) having the amino acidsequence shown in SEQ ID NO:4 (FIG. 13B); or (iii) that is an analog of(i) or (ii).
 35. A soluble fgl2 protein (i) encoded by the nucleic acidmolecule shown in SEQ ID NO:5 (FIG. 14A); (ii) having the amino acidsequence shown in SEQ ID NO:6 (FIG. 14B); or (iii) that is an analog of(i) or (ii).
 36. An isolated nucleic acid molecule encoding a solublefgl2 comprising: (a) a nucleic acid sequence as shown in FIG. 12A(SEQ.ID.NO:1); (b) a nucleic acid sequence that is complimentary to anucleic acid sequence of (a); (c) a nucleic acid sequence that hassubstantial sequence homology to a nucleic acid sequence of (a) or (b);(d) a nucleic acid sequence that is an analog of a nucleic acid sequenceof (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to anucleic acid sequence of (a), (b), (c) or (d) under stringenthybridization conditions.
 37. An isolated nucleic acid molecule encodinga soluble fgl2 comprising: (a) a nucleic acid sequence as shown in FIG.13A (SEQ.ID.NO:3); (b) a nucleic acid sequence that is complimentary toa nucleic acid sequence of (a); (c) a nucleic acid sequence that hassubstantial sequence homology to a nucleic acid sequence of (a) or (b);(d) a nucleic acid sequence that is an analog of a nucleic acid sequenceof (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to anucleic acid sequence of (a), (b), (c) or (d) under stringenthybridization conditions.
 38. An isolated nucleic acid molecule encodinga soluble fgl2 comprising: (a) a nucleic acid sequence as shown in FIG.14A (SEQ.ID.NO:5); (b) a nucleic acid sequence that is complimentary toa nucleic acid sequence of (a); (c) a nucleic acid sequence that hassubstantial sequence homology to a nucleic acid sequence of (a) or (b);(d) a nucleic acid sequence that is an analog of a nucleic acid sequenceof (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to anucleic acid sequence of (a), (b), (c) or (d) under stringenthybridization conditions.
 39. A method of diagnosing a soluble fgl2related immune suppression condition comprising: (a) incubating abiological sample from a patient with an antibody that binds to solublefgl2 under conditions that promote soluble fgl2/antibody complexformation; (b) determining the presence and/or levels of fgl2 byindirectly or directly detecting said complex formation; (c) thepresence of fgl2 and/or level thereof being indicative of an fgl2related immune suppression condition.
 40. A method according to claim 39wherein the soluble fgl2 is (1) (i) encoded by the nucleic acid moleculeshown in SEQ ID NO:1 (FIG. 12A); (ii) having the amino acid sequenceshown in SEQ ID NO:2 (FIG. 12B); or (iii) that is an analog of (i) or(ii). (2) (i) encoded by the nucleic acid molecule shown in SEQ ID NO:3(FIG. 13A); (ii) having the amino acid sequence shown in SEQ ID NO:4(FIG. 13B); or (iii) that is an analog of (i) or (ii); or (3) (i)encoded by the nucleic acid molecule shown in SEQ ID NO:5 (FIG. 14A);(ii) having the amino acid sequence shown in SEQ ID NO:6 (FIG. 14B); or(iii) that is an analog of (i) or (ii).